Method for cloning and expression of OkrAI restriction endonuclease and methyltransferase

ABSTRACT

The present invention relates to recombinant DNA encoding the OkrAI restriction endonuclease as well as OkrAI methylase, expression of OkrAI restriction endonuclease in  E. coli  cells containing the recombinant DNA.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to recombinant DNA that encodes the OkrAI restriction endonuclease (OkrAI endonuclease or OkrAI) as well as OkrAI methyltransferase (OkrAI methylase or M. OkrAI), expression of OkrAI endonuclease and methylase in E. coli cells containing the recombinant DNA.

[0002] OkrAI endonuclease is found in the strain of Oceanospirillum kriegii (ATCC 35192). It recognizes the double-stranded DNA sequence 5′G/GATCC3′ (/ indicates the cleavage position) and cleaves between two Gs to generate a 4-base 5′ overhang. OkrAI methylase is also found in the same strain, which recognizes the same DNA sequence and presumably modifies the cytosine at the N4 position on hemi-methylated or non-methylated GGATCC sites.

[0003] Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.

[0004] Restriction endonucleases recognize and bind particular sequences of nucleotides (the ‘recognition sequence’) along the DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and twenty-eight restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res.29:268-269 (2001)).

[0005] Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5′TTT/AAA3′, 5′PuG/GNCCPy3′ and 5′ CACNNN/GTG3′ respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5′G/AATTC3′.

[0006] A second component of bacterial/viral restriction-modification (R-M) systems is the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.

[0007] With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by ‘shotgun’ procedures, when they occur at frequencies as low as 10⁻³ to 10⁻⁴. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.

[0008] A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719 (1980); HhaII: Mann et al., Gene 3-:97-112 (1978); PstI: Walder et al., Proc. Sat. Acad. Sci. 78:1503-1507 (1981)). Since the expression of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.

[0009] Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676 (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406 (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509 (1985); Bsa45I: Wayne et al. Gene 202:83-88 (1997)).

[0010] A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421 (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119, (1983); and BsaI: Walder et al., J. Biol. Chem. 258:1235-1241 (1983)).

[0011] A more recent method, the “endo-blue method”, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403 (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that some positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.

[0012] There are three major groups of DNA methyltransferases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone, et al. J. Mol. Biol. 253:618-632, (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer DNA sites resistant to restriction digestion. For example, Dcm methylase modification of 5′ CCWGG3′ (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpM methylase can modify the CG dinucleotide and make the NotI site (5′GCGGCCGC3′) refractory to NotI digestion (New England Biolabs' catalog, 2000-01, page 220). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.

[0013] Type II methylase genes have been found in many sequenced bacterial genomes (GenBank, http://www.ncbi.nlm.nih.gov; and REBASE®, http://rebase.neb.com/rebase). Direct cloning and over-expression of ORFs adjacent to the methylase genes yielded restriction enzymes with novel specificities (Kong et al. Nucl. Acids Res. 28:3216-3223 (2000)). Thus microbial genome mining emerged as a new way of screening/cloning new type II restriction enzymes and methylases and their isoschizomers.

[0014] Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a strong commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes and methylases. Such over-expression strains should also simplify the task of enzyme purification.

SUMMARY OF THE INVENTION

[0015] The present invention relates to a method for cloning OkrAI restriction endonuclease gene (okrAIR which is an isoschizomer of BamHI) from Oceanospirillum kriegii into E. coli by methylase selection, inverse PCR, and direct PCR from genomic DNA using primers based on the DNA sequences obtained via methylase selection.

[0016] Initial attempts to clone OkrAI by methylase selection were unsuccessful. Specifically, two BamHI resistant clones were first identified in an EcoRI genomic DNA library. However, they turned out to be false positive since they had suffered DNA rearrangements and the DNA inserts did not encode any DNA methylase or endonuclease.

[0017] The okrAIM gene was successfully cloned and selected from an ApoI genomic DNA library by the methylase selection method. Specifically, the entire insert was digested with restriction enzyme ApoI and the resulting fragments were subcloned and sequenced. Deletion clones were also constructed to facilitate the sequencing efforts. The okrAIM gene encoding an N4 cytosine (N4C) methylase was identified within the insert. A small ORF encoding a putative control protein (okrAIC gene) was also found. Further upstream a partial ORF was found that has amino acid sequence similarity to BamHI endonuclease. Since OkrAI is an isoschizomer of BamHI, it was reasoned that they may share some amino acid sequence similarity. To obtain the entire sequence inverse PCR primers were synthesized based on the truncated gene sequence. Inverse PCR product carrying the remaining coding sequence was obtained in the AluI digested and self-ligated template. After gel purification, the PCR product was sequenced and the okrAIR gene was cloned into a T7 expression vector pAII17S. OkrAI endonuclease was over-expressed and the over-produced protein (22 kDa) was detected in SDS-PAGE. OkrAI endonuclease was also fused to an intein and a chitin binding domain so that OkrAI can be purified via affinity tag and chitin column chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1. Gene organization of OkrAI restriction-modification system. okrAIR, OkrAI restriction endonuclease gene; okrAIM, OkrAI methylase gene.

[0019]FIG. 2. OkrAI methylase gene sequence (okrAIM, 1224 bp) (SEQ ID NO:1) and its encoded amino acid sequence (SEQ ID NO:2).

[0020]FIG. 3. OkrAI endonuclease gene sequence (okrAIR, 585 bp) (SEQ ID NO:3) and its encoded amino acid sequence (SEQ ID NO:4).

[0021]FIG. 4. OkrAI C gene sequence (okrAIC, 237 bp) (SEQ ID NO:5) and its encoded amino acid sequence (SEQ ID NO:6).

[0022]FIG. 5. Purified recombinant OkrAI endonuclease analyzed on SDS-PAGE. Lane 1, protein size marker; Lanes 3 to 6, 40, 20, 5, and 1 μg OkrAI endonuclease protein, respectively. The apparent molecular mass of OkrAI is 22 kDa.

[0023]FIG. 6. OkrAI endonuclease activity assay on λ DNA in the presence of 1 to 10% glycerol. Lane 1, λ DNA; lanes 2 to 11, 1% to 10% of glycerol; lane 12, DNA size marker. Assay condition: 100 units of OkrAI to digest 1 μg λ DNA in 1× NEB buffer 3 at room temperature (r.t.) for 1 h.

[0024]FIG. 7. OkrAI activity assay following heat-treatment. Lane 1, λ DNA; lane 2, 1 μg λ DNA digested with 20 units of OkrAI; lane 3, 1 μ g λ DNA treated with heat-treated OkrAI (20 units); lane 4, 1 μg λ DNA digested with 100 units of okrAI; lane 5, 1 μg λ DNA treated with heat-treated OkrAI (100 units). Assay condition: OkrAI or heat-treated OkrAI to digest 1 μg λ DNA in 1× NEB buffer 3 at r.t. for 1 h in a total volume of 50 μl. 20 and 100 units of OkrAI were first heated at 65° C. for 30 min.

DETAILED DESCRIPTION OF THE INVENTION

[0025] 1. Preparation of Genomic DNA, Restriction Digestion, and Construction of Genomic DNA Library

[0026] Genomic DNA was prepared from Oceanospirillum kriegii by the standard method. ApoI endonuclease was used to partially digest the genomic DNA. ApoI fragments in 2-10 kb were purified and ligated to EcoRI digested and CIP treated pRRS vector that contains multiple OkrAI sites. The ligated DNA was used to transform ER2502 by electroporation. Approximately 10,000 Ap^(R) transformants were derived from the transformation. All the colonies were pooled and amplified. Plasmid DNA was prepared to generate a plasmid DNA library.

[0027] 2. Cloning of okrAIM Gene by Methylase Selection

[0028] The primary plasmid library was challenged with BamHI. The digested DNA was transferred into ER2502 by transformation, resulting in ˜100 Ap^(R) survivors. Plasmid DNA was prepared from cultures of the Ap^(R) survivors. Following BamHI digestion and agarose gel electrophoresis one resistant clone was detected.

[0029] 3. Restriction Mapping and Subcloning of the Insert

[0030] The resistant plasmid DNA was digested with a number of restriction enzymes to estimate the insert size. The insert size was estimated to be ˜2.8 kb. Multiple ApoI fragments from the insert were gel-purified and subcloned into pUC19. The inserts were sequenced using pUC universal primers. HincII and HindIII fragment deletion clones were also constructed and sequenced. One ORF has high homology to the N4C methylase family and this ORF was named okrAIM gene. A second ORF has sequence homology to the family of R-M system control genes (okrAIC gene). One truncated ORF (˜400 bp) was also found. This partial ORF was a candidate for the okrAIR gene.

[0031] 4. Inverse PCR Amplification of DNA Upstream of okrAIM and okrAIC Gene

[0032] After identification of the methylase gene, efforts were made to clone adjacent upstream DNA. Inverse PCR primers were made based on the known truncated sequence.

[0033] The genomic DNA was digested with 4-base cutting restriction enzymes, purified, and self-ligated. The circular DNA molecules were used as templates for inverse PCR. PCR product was found using the AluI digested and ligated template. The PCR product was purified and sequenced directly with the primers for the inverse PCR reaction. The AluI fragment generated about 400 bp new DNA sequence. A 585-bp ORF was found and named the okrAIR gene. Transcription of M and R genes is oriented in the same direction (see FIG. 1 for gene organization).

[0034] 5. Expression of okrAIR Gene in E. coli

[0035] The non-cognate methylase, M. BamHI, was used to pre-modify E. coli host ER2566 for OkrAI endonuclease expression. The okrAIR gene was cloned in a T7 expression vector pAII17S. The okrAIR gene was amplified by PCR, digested with NdeI and SalI and ligated to pAII17 with compatible ends, and transformed into the M. BamHI modified host ER2566. Alternatively, the okrAIR gene was amplified in PCR and digested with NdeI and ligated to NdeI and SmaI digested pTYB2 expression vector. In pTYB2 the okrAIR gene is fused to an intein from Saccharomyces cerevisiae VMA1 gene and chitin binding domain. Therefore OkrAI endonuclease can be purified by chitin column chromatography. After the fusion protein is bound to the chitin column, the OkrAI endonuclease can be cleaved from the fusion by addition of DTT. The endonuclease gene insert in pAII17S or pTYB2 was sequenced and confirmed to be the wild-type coding sequence. The final yield from the T7 expression system, ER2566 [pACYC-BamHIM, pAII17S-OkrAIR], is approximately 5×10⁶ units per gram of wet IPTG-induced cells.

[0036] The present invention is further illustrated by the following Examples. These Examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof.

[0037] The references cited above and below are herein incorporated by reference.

EXAMPLE 1 Cloning of OkrAI Restriction-Modification System in E. coli

[0038] 1. Preparation of Genomic DNA

[0039] Genomic DNA was prepared from Oceanospirillum kriegii (ATCC 35192) by the standard procedure consisting of the following steps:

[0040] a. Cell lysis by addition of lysozyme (2 mg/ml final), sucrose (1% final), and 50 mM Tris-HCl, pH 8.0.

[0041] b. Further cell lysis by addition of SDS at a final concentration of 0.1%.

[0042] c. Further cell lysis by addition of 1% Triton X-100, 62 mM EDTA, 50 mM Tris-HCl, pH 8.0.

[0043] d. Removal of proteins by phenol-CHCl₃ extraction of DNA 3 times (equal volume) and CHCl₃ extraction once.

[0044] e. Dialysis in 4 liters of TE buffer, buffer change twice.

[0045] f. RNase A treatment to remove RNA.

[0046] g. Genomic DNA precipitation in 95% ethanol, centrifuged, washed, dried and resuspended in TE buffer.

[0047] 2. Restriction Digestion of Genomic DNA and Construction of Genomic DNA Library

[0048] EcoRI Complete Library:

[0049] EcoRI digested genomic DNA was ligated to EcoRI cut and CIP treated pEZL38 vector DNA (pEZL38 carries two OkrAI sites) at 16° C. overnight. The ligated DNA was transferred into ER2267 by transformation. Transformants were plated on Ap plates overnight at 37° C. Approximately 22,00 Ap^(R) colonies were pooled. Plasmids were purified from the cells by alkaline lysis method. Five ug of plasmid were digested with 3 units or 30 unitps of BamHI at 37° C. overnight. The challenged DNA was used to transform ER2677. Ap^(R) survivor transformants were amplified in small cultures and plasmids were purified from individual cultures and digested with Ba HI to test resistance to BamHI digestion. Two resistant clones (#18 and #21) were identified and later proved to be false positive. These two clones do not display M. OkrAI and OkrAI endonuclease activities.

[0050] ApoI Partial Library:

[0051] Varying units of restriction enzymes ApoI were used to digest 5 μg genomic DNA (ApoI: 2, 1, 0.5, 0.25, 0.125 units) at 50° C. for 1 h to achieve limited partial digestion. It was found that 0.5 and 0.25 units of ApoI gave rise to optimal partial digestion. The partially digested DNA in the range of 2 kb to 10 kb was gel-purified from a low-melting agarose gel. Following β-agarase treatment and ethanol precipitation, the ApoI digested DNA was ligated to EcoRI and CIP treated pRRS vector that contains multiple OkrAI sites. The vector pRRS is a pUC-based high-copy-number plasmid for cloning and expression of genes in E. coli. The ligated DNA was used to transform EndA⁻ RR1 competent cells (ER2502, New England Biolabs, Inc. collection (Beverly, Mass.)) by electroporation. Approximately 10,000 Ap^(R) transformants were obtained for the partial library. All the colonies were pooled and amplified in 1 liter LB+Ap (100 μg/ml) overnight. Plasmid DNA was prepared by Qiagen (Studio City, Calif.) Maxi-prep columns, generating a plasmid DNA library.

[0052] 3. Cloning of OkrAIM Gene by Methylase Selection

[0053] The primary plasmid DNA library (1 μg, 2 μg, and 5 μg DNA) was challenged with 100 units of BamHI at 37° C. overnight in a total volume of 100 l in 1× BamHI buffer (150 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT). Because BamHI and OkrAI are isoschizomer, it was predicted that M. OkrAI modified plasmid DNA might be resistant to BamHI digestion. The BamHI digested DNA was transferred into ER2502 by transformation, generating ˜100 Ap^(R) survivors. Plasmid DNA was prepared by Qiagen (Studio City, Calif.) spin columns from 36 overnight cell cultures (1.5 ml×36). After digestion of the plasmid DNA by BamHI, one complete resistant clone (#30) was detected and it was further characterized.

[0054] 4. Restriction Mapping and Subcloning of the Insert

[0055] The resistant plasmid DNA was digested with restriction enzymes ApoI, BamHI, EcoRI, HindIII, PvuII, PstI, SacI, and SphI to estimate the insert size. The insert size was estimated to be ˜2.8 kb. The BamHI-resistant plasmid was digested with ApoI and four ApoI fragments were gel-purified from a low-melting agarose gel and sub-cloned into EcoRI digested and CIP treated pUC19. The ApoI fragment inserts were screened and sequenced using pUC forward and reverse universal primers.

[0056] Two deletion clones were also constructed. HincII fragment and HindIII fragment deletion clones were also constructed by HincII or HindIII digestion of the resistant clone and self-ligation at a low DNA concentration. After retransformation and confirmation of each deletion, the deletion clones were also sequenced by pUC primers and custom made primers. DNA sequencing was performed using the dye terminator sequencing kit from PE Biosystems (Foster City, Calif.). The sequencing primers have the following sequences: 5′TGCATTAACAGGACTTCAATCACC3′ (SEQ ID NO: 7) 5′ACATAATGCAGGCCACGCCCAACC3′ (SEQ ID NO: 8) 5′CGGTCTGGATGAAAGGAATTCGGC3′ (SEQ ID NO: 9) 5′AATCTTTCAAATCGCCGTAGCACT3′ (SEQ ID NO: 10)

[0057] One open reading frame (ORF) has amino acid sequence homology to the N4 cytosine methylase family and BamHI methylase. This ORF was named the okrAIM gene. A second ORF has sequence homology to the control gene (C gene) of some restriction-modification systems. One truncated ORF (˜400 bp) was also found. This partial ORF was a candidate for the okrAIR gene. Therefore, major efforts were made to clone the remaining part of the truncated ORF.

[0058] 5. Inverse PCR Amplification of DNA Downstream of OkrAI Methylase

[0059] After identification of the methylase gene, it is desirable to clone the adjacent genes in order to find the cognate endonuclease gene. The genomic DNA was digested with 4-base cutting restriction enzymes AluI, HhaI, NlaIII, RsaI, Sau3AI, and TaqI, respectively in appropriate restriction buffers and temperatures. TaqI digestion was performed at 65° C. and the rest at 37° C.

[0060] The digested DNA was purified through Qiagen (Studio City, Calif.) spin columns. DNA self-ligation was performed at a low DNA concentration (2 μg/ml) overnight at 16° C. T4 DNA ligase was inactivated at 65° C. for 30 min and the circular DNA was precipitated in ethanol and used as the template for inverse PCR. Two primers were synthesized with the following sequences: 5′TCCTGTATGAGACATCTTCATCA (184-32) (SEQ ID NO: 11) G3′ 5′CACCATTACCTTTGCGAACAGGA (184-33) (SEQ ID NO: 12) T3′

[0061] PCR conditions were 95° C. for 1 min, 50° C. for 1 min, 72° C. for 2 min for 40 cycles. A PCR product (˜450-500 bp) was found in the AluI template. It was purified from a low-melting agarose gel, treated with β-agarase for 2 h, precipitated with ethanol, resuspended in TE buffer, and sequenced directly with primers 184-32 and 184-33. Sequencing of the AluI fragment generated ˜400 bp new DNA sequence. A start codon and a stop codon were found in the ORF and this ORF was named the okrAIR gene (gene size 585 bp). Transcription of R and M genes is oriented in the same direction (see FIG. 1 for gene organization). The orientation of the okrAIC gene is opposite of the okrAIR gene.

EXAMPLE 2 Expression of okrAIR Gene in E. coli Using T7 Expression System (pAII17S Vector)

[0062] Two PCR primers were synthesized for the amplification of the okrAIR gene. The primers have the following sequences: 5′GGAGGAGTCCATATGAAAATAA (190-121) (SEQ ID NO: 13) AGCGTATTGAGGTCCTTATA3′ 5′GGAGGAGTCGACTCACCTTATA (190-122) (SEQ ID NO: 14) GCACGACCATCTGTACCCTT 3′

[0063] An NdeI site and a SalI site were incorporated in the forward and reverse primers, respectively. The okrAIR gene was amplified from the genomic DNA in a PCR reaction under the conditions: 95° C. for 1 min, 60° C. for 1 min, 72° C. for 1 min for 20 cycles; 2 units of Vent® DNA polymerase with varying concentrations of Mg++ (2 to 10 mM) in a total volume of 100 μl. The PCR DNA was purified by phenol-chloroform extraction and chloroform extraction and ethanol precipitation. It was then digested with restriction enzymes NdeI and SalI for 3 h at 37° C. The DNA was purified further by phenol-chloroform extraction and chloroform extraction and ethanol precipitation. The PCR DNA was ligated to a modified T7 expression vector pAII17S. Vector pAII17S was derived from pAII17. The BamHI site in pAII17 was filled-in with Klenow fragment and a SalI linker was inserted to replace the BamHI site (Xu, S.-Y. New England Biolabs, Beverly, Mass.). The cloning sites in pAII17S are NdeI and SalI sites. Ligated PCR DNA/vector pAII17S were transferred into pre-modified expression host ER2566 [pACYC184-bamHIM] by transformation. Thirty-six transformants were screened, but none of them contained the desired okrAIR gene insert. To increase the cloning efficiency, the PCR DNA was digested again with NdeI and SalI overnight in 1× NEB buffer 4 (>16 h). The PCR DNA was purified and ligated to pAII17S with compatible ends. The ligated DNA was then transferred into E. coli host ER2566 [pACYC184-bamHIM]. This time after screening 18 transformants, 17 clones with inserts were found. Ten ml of cell cultures were grown for 10 isolates to late log phase and IPTG was added to a final concentration of 0.5 mM to induce OkrAI production. IPTG induction continued for 3 h and cells were collected by centrifugation. Cell pellet was resupended in a sonication buffer (50 mM Tris-HCl, pH 7.8, 10 mM β-mercaptoethanol, 50 mM NaCl) and cell lysis was completed by sonication. Cell debris was removed by centrifugation and clarified cell lysate was used to assay OkrAI endonuclease activity on λ DNA substrate. All ten isolates displayed OkrAI endonuclease activity in cell extracts. Isolate #9 was further characterized in a stability study in 1 L culture. One ml of cells were inoculated into 1 L of LB+Ap+Cm and cultured overnight in a shaker at 37° C. Twenty ml of the overnight cells were inoculated into a fresh 1 L LB+Ap+Cm and shaken at 37° C. for 4 h to late log phase. IPTG was added to final concentration of 0.5 mM for 3 h. Cells were harvested and lysed by sonication and cell extract was assayed again for OkrAI activity λ DNA substrate. High OkrAI activity was detected following a thousand-fold dilution of the cell extract. The OkrAI activity yield is approximately 5×10⁶ units per gram of wet cells.

[0064] The plasmid DNA pAII17S-okrAIR was prepared by Qiagen (Studio City, Calif.) tip-20 column and the entire insert was sequenced. It was found that the insert contained the okrAIR wild-type coding sequence.

[0065] The strain NEB#1164 ER2566 [pACYC184-bamHIM, pAII17S-okrAIR] has been deposited under the terms and conditions of the Budapest Treaty with the American Type Culture Collection on Feb. 12, 2003 and received ATCC Accession No. PTA-5002.

EXAMPLE 3 Expression of OkrAI with an Intein and Chitin Binding Protein Fusion in pTYB2

[0066] Two PCR primers were synthesized for the amplification of the okrAIR gene. The primers have the following sequences: 5′GGAGGAGTCCATATGAAAATAA (190-121) (SEQ ID NO: 15) AGCGTATTGAGGTCCTTATA3′ 5′CCTTATAGCACGACCATCTGTA (191-21r) (SEQ ID NO: 16) CCCTT3′

[0067] An NdeI site was incorporated in the forward primer. OkrAIR gene was amplified from the genomic DNA in a PCR reaction under the conditions: 95° C. for 1 min, 60° C. for 1 min, 72° C. for 1 min for 20 cycles; 2 units of Vent® DNA polymerase with varying concentration of Mg⁺⁺ (2 to 10 mM) in a total volume of 100 μl. The PCR DNA was purified by phenol-chloroform extraction and chloroform extraction and ethanol precipitation. It was then digested with restriction enzyme NdeI for 3 h at 37° C. The DNA was purified further by phenol-chloroform extraction and chloroform extraction and ethanol precipitation. The vector DNA pTYB2 was digested with NdeI and SmaI for 3 h at 37° C. and further purified by phenol-chloroform extraction and chloroform extraction and ethanol precipitation. The PCR DNA (one NdeI end and one blunt end) was ligated to NdeI and SmaI digested pTYB2 and the ligated DNA was transferred into expression host ER2566 [pACYC184-bamHIM] by transformation. After screening 72 plasmids isolated from cultures of the transformants, eight clones were found to contain the desired PCR insert. These 8 clones with inserts were grown in 10 ml LB+Ap (50 μg/ml) and Cm (33 μg/ml) to late log phase. IPTG was added to a final concentration of 0.5 mM to induce OkrAI-intein-CBD fusion protein production. IPTG induction continued for 3 h and cells were collected by centrifugation. Cell pellet was resupended in a sonication buffer (50 mM Tris-HCl, pH 7.8, 10 mM β-mercaptoethanol, 50 mM NaCl) and cell lysis was completed by sonication. Cell debris was removed by centrifugation and the clarified cell lysate was incubated with DTT and used to assay OkrAI endonuclease activity on λ DNA substrate. The addition of DTT facilitates the cleavage of OkrAI from the fusion protein. Four isolates (#11, #13, #14, and #26) displayed high OkrAI endonuclease activity in cell extracts. Plasmid DNA pTYB2-okrAIR was prepared by Qiagen (Studio City, Calif.) tip-20 column and the entire insert was sequenced. It was found that each insert contained the wild-type coding sequence, except for the inclusion of a Gly codon at the C-terminal fusion junction. The intein-CBD-OkrAI fusion expression strain is ER2566 [pACYC184-bamHIM, pTYB2-okrAIR].

EXAMPLE 4 Large Scale Purification of OkrAI Endonuclease

[0068] All activity determinations were done by incubating OkrAI fractions with lambda DNA for specified times at 37° C.

[0069] A wet cell mass of 115 grams was suspended at 4° C. in buffer A (100 mM NaCl, 20 mM KPO4, (pH 6.8), 1 mM DTT, 1 mM EDTA). All subsequent procedures were done at 4° C. The cells were sonicated and the debris removed by centrifugation at 20,000× g for 1 hour.

[0070] The supernatant was applied to a 5×15 cm phosphocellulose column (Whatman P11) equilibrated in buffer A. After washing to remove unbound material, the activity was eluted with a 2000 ml gradient from 0.1 to 2 M NaCl. OkrAI activity eluted at approximately 0.9 M NaCl.

[0071] The peak tubes were combined and loaded directly onto a 5×6 cm hydroxylapatite (Bio-Rad Laboratories, (Richmond, Calif.)) column. After washing with buffer A to remove unbound material, it was determined that the OkrAI activity was in the flow-through and wash for the column. These fractions were combined and dialyzed against buffer B (100 mM NaCl, 10 mM Tris-HCl (pH 7.8), 1 mM DTT, 1 mM EDTA).

[0072] The dialyzed material was loaded onto a 5×14 cm DEAE column (Pharmacia (Milwaukee, Wis.)). After washing the column with buffer B, it was determined that the OkrAI activity was again in the flow-through and wash for the column.

[0073] These fractions were combined, diluted with buffer to lower the NaCl concentration to 50 mM NaCl and change the pH to 7.3, and loaded directly onto a 5×19 cm heparin column (Pharmacia (Milwaukee, Wis.)). After washing with buffer C, (50 mM NaCl, 10 mM Tris-HCl (pH 7.3), 1 mM DTT, 1 mM EDTA), the activity was eluted with a 2000 ml gradient from 50 mM to 1 M NaCl. OkrAI activity eluted at approximately 0.7 M NaCl. These fractions were combined and dialyzed against buffer B.

[0074] The dialyzed material was loaded onto a 2.5×10 cm Q-Sepharose (Pharmacia (Milwaukee, Wis.)) column. After washing with buffer B, it was determined that the OkrAI activity was in the flow-through and wash for the column.

[0075] The fractions were combined and loaded directly onto a 2.5×9 cm Affi-gel Blue (Bio-Rad Laboratories (Richmond, Calif.)) column. After washing the column with buffer B, the activity was eluted with a 500 ml gradient from 50 mM to 2 M NaCl. The activity eluted at a salt concentration of approximately 0.9 M NaCl. These fractions were combined and dialyzed against buffer C (150 mM NaCl, 10 mM Tris-HCl (pH 7.3), 1 mM DTT, 0.1 mM EDTA).

[0076] The dialyzed material was loaded onto a 1×4.5 cm Heparin (Pharmacia (Milwaukee, Wis.)) column. After washing the column with buffer C, the activity was eluted with a 200 ml gradient from 0.15M to 0.8M NaCl. The activity eluted at a salt concentration of approximately 0.5M. The fractions containing activity were dialyzed against buffer D (100 mM NaCl, 20 mM KPO4 (pH 6.8), 1 mM DTT, 0.1 mM EDTA)

[0077] The dialyzed material was loaded onto a 1×4 cm phosphocellulose (Whatman P11) column. After washing the column with buffer D, the activity was eluted with a 200 ml gradient from 0.1 M NaCl to 1 M NaCl. The activity eluted at a salt concentration of approximately 0.5 M NaCl.

[0078] The fractions containing activity were collected and dialyzed against buffer E (75 mM NaCl, 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA). After some precipitation was noted, the NaCl concentration was increased to 200 mM, the tris concentration was changed to 20 mM Tris-HCl (pH 8) 1 mM DTT and 5% (v/v) glycerol were added. Some remaining precipitated material was removed by centrifugation and 0.2 micron filtration.

[0079] This final material was evaluated on SDS-PAGE (see FIG. 5) and by standard quality control assays and used for subsequent activity assays.

EXAMPLE 5 The Use of Room Temperature Incubation to Reduce OkrAI “Star” Activity

[0080] OkrAI displayed “star” activity at high glycerol concentration and prolonged incubation, a property similar to BamHI endonuclease. However, it was found that room temperature (r.t.) incubation dramatically reduced the OkrAI “star” activity. Room temperature (25° C.) reduced OkrAI specific activity only 2-fold, but the “star” activity was much reduced. In a 50 μl reaction volume, 20 units to 800 units OkrAI digestion of 1 μg of λ DNA did not show any “star” site cleavage at r.t. for 1 h.

[0081] OkrAI “star” activity was also tested in the presence of 1-10% glycerol. The reaction conditions were: 100 units of OkrAI, 1× buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT), 1 μg of λ DNA, glycerol 1% to 10%, in a total volume of 50 μl at r.t. for 1 h. OkrAI does not display any “star” activity in the presence of 1% to 10% of glycerol at r.t. (see FIG. 6, lanes 2 to 11).

[0082] OkrAI endonuclease can be heat-inactivated at 65° C. for 30 min. 20 units and 100 units of OkrAI were first heated at 65° C for 30 min. Then they were incubated with 1 μg of λ DNA in 1× buffer 3 at r.t. for 1 h. After the heat treatment, no OkrAI endonuclease activity was detected, indicating that OkrAI was completely inactivated (see FIG. 7, lanes 3 and 5).

[0083] It was concluded that OkrAI displays no “star” activity at r.t. in the presence of 1-10% of glycerol (100 units of OkrAI tested). OkrAI does not show detectable “star” activity in over-digestion (20 to 800 units to digest 1 μg DNA). 100 units of OkrAI can be heat-inactivated at 65° C. for 30 min. The heat-inactivation property provides more convenience for molecular biology applications.

1 16 1 1224 DNA Oceanospirillum kriegii CDS (1)..(1224) 1 atg ggc tat tca gac cac ttt ttt aaa gct ttg aat ata tcg gtt gaa 48 Met Gly Tyr Ser Asp His Phe Phe Lys Ala Leu Asn Ile Ser Val Glu 1 5 10 15 gac agg aaa agt ttg tct gag ttt tcc aaa aga agc ggt ata cct gtt 96 Asp Arg Lys Ser Leu Ser Glu Phe Ser Lys Arg Ser Gly Ile Pro Val 20 25 30 aag aaa ttg aag tac tac aac gaa gga aat gta gta cct acc ggc aag 144 Lys Lys Leu Lys Tyr Tyr Asn Glu Gly Asn Val Val Pro Thr Gly Lys 35 40 45 gac ctg gaa aag ata ctc tct acc gcg aac ctt tct gag gtt ttg ctt 192 Asp Leu Glu Lys Ile Leu Ser Thr Ala Asn Leu Ser Glu Val Leu Leu 50 55 60 cgc ttg aaa atg ggt agg ctg gat aag gat att cta gcg gca ata cag 240 Arg Leu Lys Met Gly Arg Leu Asp Lys Asp Ile Leu Ala Ala Ile Gln 65 70 75 80 gaa aat gcg gaa agt gtt ctt gct gaa atc gac ggt ttt aat ccg gtt 288 Glu Asn Ala Glu Ser Val Leu Ala Glu Ile Asp Gly Phe Asn Pro Val 85 90 95 gtg gat tct ccg gag gtc gac tgt aca ttg gag ttt gaa acc aga ctc 336 Val Asp Ser Pro Glu Val Asp Cys Thr Leu Glu Phe Glu Thr Arg Leu 100 105 110 ggt aaa ctt tat cgt ggg gat tgt tat tct cta ctg aag tca atg gaa 384 Gly Lys Leu Tyr Arg Gly Asp Cys Tyr Ser Leu Leu Lys Ser Met Glu 115 120 125 agc gat tct gtt gat ctg ata ttc tct gat ccc cct ttt aat ctt gac 432 Ser Asp Ser Val Asp Leu Ile Phe Ser Asp Pro Pro Phe Asn Leu Asp 130 135 140 aag ata tat cct tct gat atg gat gac aat ata aag gtg gat aag tat 480 Lys Ile Tyr Pro Ser Asp Met Asp Asp Asn Ile Lys Val Asp Lys Tyr 145 150 155 160 att ggc tgg agt cag gag tgg ata aag gaa tgc gct cgt gtt tta aag 528 Ile Gly Trp Ser Gln Glu Trp Ile Lys Glu Cys Ala Arg Val Leu Lys 165 170 175 cct ggt ggt gcg ctt ttc atg tgg aac ctc ccg aag tgg aat gtg gca 576 Pro Gly Gly Ala Leu Phe Met Trp Asn Leu Pro Lys Trp Asn Val Ala 180 185 190 tta ggt tcg ttt gtt gat ggc ctg ctt acg ttc aga aac tgg att ggc 624 Leu Gly Ser Phe Val Asp Gly Leu Leu Thr Phe Arg Asn Trp Ile Gly 195 200 205 gta gac ata aaa tat agc ctt cca att aga aat cga ttg tat cca tct 672 Val Asp Ile Lys Tyr Ser Leu Pro Ile Arg Asn Arg Leu Tyr Pro Ser 210 215 220 cat tat tcg ttg atg tat tac atc aag ggt gaa aag ccg aat tcc ttt 720 His Tyr Ser Leu Met Tyr Tyr Ile Lys Gly Glu Lys Pro Asn Ser Phe 225 230 235 240 cat cca gac cgt ttg gct atg gat gtt tgc cca aag tgc tac ggc gat 768 His Pro Asp Arg Leu Ala Met Asp Val Cys Pro Lys Cys Tyr Gly Asp 245 250 255 ttg aaa gat tat ggc ggt tac aag gat aag atg aat ccg ttg ggt att 816 Leu Lys Asp Tyr Gly Gly Tyr Lys Asp Lys Met Asn Pro Leu Gly Ile 260 265 270 aat ctt tct gat gtc tgg tat gac att cct cct gta agg cat gca aag 864 Asn Leu Ser Asp Val Trp Tyr Asp Ile Pro Pro Val Arg His Ala Lys 275 280 285 tac aaa agg aga aag ggc tcc aat gag ctt tcg tta aag ctg ttg gac 912 Tyr Lys Arg Arg Lys Gly Ser Asn Glu Leu Ser Leu Lys Leu Leu Asp 290 295 300 agg atc att gag atg gct tca gac gaa ggt gat ttg gtg ttt gat cca 960 Arg Ile Ile Glu Met Ala Ser Asp Glu Gly Asp Leu Val Phe Asp Pro 305 310 315 320 ttc ggg ggc tcc ggc aca acg tat atg gca gcc gag cta aag ggc cgg 1008 Phe Gly Gly Ser Gly Thr Thr Tyr Met Ala Ala Glu Leu Lys Gly Arg 325 330 335 aga tgg gtt ggc tgt gaa ctg gga cca aca gat att att aaa gag cga 1056 Arg Trp Val Gly Cys Glu Leu Gly Pro Thr Asp Ile Ile Lys Glu Arg 340 345 350 ttt tct ttg atc gaa gaa gaa agg gat ata ctc aat ggt tat cga ggg 1104 Phe Ser Leu Ile Glu Glu Glu Arg Asp Ile Leu Asn Gly Tyr Arg Gly 355 360 365 cga gta aat gct ctt ttc cct gag aaa acc aga tcc gag cga gaa aaa 1152 Arg Val Asn Ala Leu Phe Pro Glu Lys Thr Arg Ser Glu Arg Glu Lys 370 375 380 cgt ggt ttg tgg act tgt gag act ttt agc aaa aac gaa cag tcg gaa 1200 Arg Gly Leu Trp Thr Cys Glu Thr Phe Ser Lys Asn Glu Gln Ser Glu 385 390 395 400 ctc ttt gac aaa aac ctg aag taa 1224 Leu Phe Asp Lys Asn Leu Lys 405 2 407 PRT Oceanospirillum kriegii 2 Met Gly Tyr Ser Asp His Phe Phe Lys Ala Leu Asn Ile Ser Val Glu 1 5 10 15 Asp Arg Lys Ser Leu Ser Glu Phe Ser Lys Arg Ser Gly Ile Pro Val 20 25 30 Lys Lys Leu Lys Tyr Tyr Asn Glu Gly Asn Val Val Pro Thr Gly Lys 35 40 45 Asp Leu Glu Lys Ile Leu Ser Thr Ala Asn Leu Ser Glu Val Leu Leu 50 55 60 Arg Leu Lys Met Gly Arg Leu Asp Lys Asp Ile Leu Ala Ala Ile Gln 65 70 75 80 Glu Asn Ala Glu Ser Val Leu Ala Glu Ile Asp Gly Phe Asn Pro Val 85 90 95 Val Asp Ser Pro Glu Val Asp Cys Thr Leu Glu Phe Glu Thr Arg Leu 100 105 110 Gly Lys Leu Tyr Arg Gly Asp Cys Tyr Ser Leu Leu Lys Ser Met Glu 115 120 125 Ser Asp Ser Val Asp Leu Ile Phe Ser Asp Pro Pro Phe Asn Leu Asp 130 135 140 Lys Ile Tyr Pro Ser Asp Met Asp Asp Asn Ile Lys Val Asp Lys Tyr 145 150 155 160 Ile Gly Trp Ser Gln Glu Trp Ile Lys Glu Cys Ala Arg Val Leu Lys 165 170 175 Pro Gly Gly Ala Leu Phe Met Trp Asn Leu Pro Lys Trp Asn Val Ala 180 185 190 Leu Gly Ser Phe Val Asp Gly Leu Leu Thr Phe Arg Asn Trp Ile Gly 195 200 205 Val Asp Ile Lys Tyr Ser Leu Pro Ile Arg Asn Arg Leu Tyr Pro Ser 210 215 220 His Tyr Ser Leu Met Tyr Tyr Ile Lys Gly Glu Lys Pro Asn Ser Phe 225 230 235 240 His Pro Asp Arg Leu Ala Met Asp Val Cys Pro Lys Cys Tyr Gly Asp 245 250 255 Leu Lys Asp Tyr Gly Gly Tyr Lys Asp Lys Met Asn Pro Leu Gly Ile 260 265 270 Asn Leu Ser Asp Val Trp Tyr Asp Ile Pro Pro Val Arg His Ala Lys 275 280 285 Tyr Lys Arg Arg Lys Gly Ser Asn Glu Leu Ser Leu Lys Leu Leu Asp 290 295 300 Arg Ile Ile Glu Met Ala Ser Asp Glu Gly Asp Leu Val Phe Asp Pro 305 310 315 320 Phe Gly Gly Ser Gly Thr Thr Tyr Met Ala Ala Glu Leu Lys Gly Arg 325 330 335 Arg Trp Val Gly Cys Glu Leu Gly Pro Thr Asp Ile Ile Lys Glu Arg 340 345 350 Phe Ser Leu Ile Glu Glu Glu Arg Asp Ile Leu Asn Gly Tyr Arg Gly 355 360 365 Arg Val Asn Ala Leu Phe Pro Glu Lys Thr Arg Ser Glu Arg Glu Lys 370 375 380 Arg Gly Leu Trp Thr Cys Glu Thr Phe Ser Lys Asn Glu Gln Ser Glu 385 390 395 400 Leu Phe Asp Lys Asn Leu Lys 405 3 585 DNA Oceanospirillum kriegii CDS (1)..(585) 3 gtg aaa ata aag cgt att gag gtc ctt ata aat aat gga tcg gtt cca 48 Val Lys Ile Lys Arg Ile Glu Val Leu Ile Asn Asn Gly Ser Val Pro 1 5 10 15 ggg att cct atg atc ttg aat gaa att caa gat gcg ata aaa aca gtt 96 Gly Ile Pro Met Ile Leu Asn Glu Ile Gln Asp Ala Ile Lys Thr Val 20 25 30 tct tgg cca gaa ggt aat aat tca ttc gtt att aat cct gtt cgc aaa 144 Ser Trp Pro Glu Gly Asn Asn Ser Phe Val Ile Asn Pro Val Arg Lys 35 40 45 ggt aat ggt gtt aaa cca att aaa aat tcc tgt atg aga cat ctt cat 192 Gly Asn Gly Val Lys Pro Ile Lys Asn Ser Cys Met Arg His Leu His 50 55 60 cag aaa ggc tgg gct ctt gaa cat cct gtt aga att aag gct gaa atg 240 Gln Lys Gly Trp Ala Leu Glu His Pro Val Arg Ile Lys Ala Glu Met 65 70 75 80 agg ccg ggc cca ttg gat gcg gtg aag atg att gga ggg aaa gca ttc 288 Arg Pro Gly Pro Leu Asp Ala Val Lys Met Ile Gly Gly Lys Ala Phe 85 90 95 gca ctt gag tgg gag acg ggg aat ata tca tcg tcg cat agg gca att 336 Ala Leu Glu Trp Glu Thr Gly Asn Ile Ser Ser Ser His Arg Ala Ile 100 105 110 aat aaa atg gtc atg ggg atg ttg gaa cgt gtg att atc gga ggt gtt 384 Asn Lys Met Val Met Gly Met Leu Glu Arg Val Ile Ile Gly Gly Val 115 120 125 ttg att ctt cca tca agg gat atg tac aac tac ttg act gat agg gta 432 Leu Ile Leu Pro Ser Arg Asp Met Tyr Asn Tyr Leu Thr Asp Arg Val 130 135 140 ggt aat ttt aga gag ctg gaa cct tat ttc tca gtt tgg cgg cag ttt 480 Gly Asn Phe Arg Glu Leu Glu Pro Tyr Phe Ser Val Trp Arg Gln Phe 145 150 155 160 aat ttg aaa gat gct tat ctt gct att gtt gaa att gaa cat gat agt 528 Asn Leu Lys Asp Ala Tyr Leu Ala Ile Val Glu Ile Glu His Asp Ser 165 170 175 gtc gat gcg cag gtt tca tta att cct aag ggt aca gat ggt cgt gct 576 Val Asp Ala Gln Val Ser Leu Ile Pro Lys Gly Thr Asp Gly Arg Ala 180 185 190 ata agg tga 585 Ile Arg 4 194 PRT Oceanospirillum kriegii 4 Val Lys Ile Lys Arg Ile Glu Val Leu Ile Asn Asn Gly Ser Val Pro 1 5 10 15 Gly Ile Pro Met Ile Leu Asn Glu Ile Gln Asp Ala Ile Lys Thr Val 20 25 30 Ser Trp Pro Glu Gly Asn Asn Ser Phe Val Ile Asn Pro Val Arg Lys 35 40 45 Gly Asn Gly Val Lys Pro Ile Lys Asn Ser Cys Met Arg His Leu His 50 55 60 Gln Lys Gly Trp Ala Leu Glu His Pro Val Arg Ile Lys Ala Glu Met 65 70 75 80 Arg Pro Gly Pro Leu Asp Ala Val Lys Met Ile Gly Gly Lys Ala Phe 85 90 95 Ala Leu Glu Trp Glu Thr Gly Asn Ile Ser Ser Ser His Arg Ala Ile 100 105 110 Asn Lys Met Val Met Gly Met Leu Glu Arg Val Ile Ile Gly Gly Val 115 120 125 Leu Ile Leu Pro Ser Arg Asp Met Tyr Asn Tyr Leu Thr Asp Arg Val 130 135 140 Gly Asn Phe Arg Glu Leu Glu Pro Tyr Phe Ser Val Trp Arg Gln Phe 145 150 155 160 Asn Leu Lys Asp Ala Tyr Leu Ala Ile Val Glu Ile Glu His Asp Ser 165 170 175 Val Asp Ala Gln Val Ser Leu Ile Pro Lys Gly Thr Asp Gly Arg Ala 180 185 190 Ile Arg 5 237 DNA Oceanospirillum kriegii CDS (1)..(237) 5 atg aaa gtg gaa tgt gca ttt gga aga atc cta aag cag ctt aga aca 48 Met Lys Val Glu Cys Ala Phe Gly Arg Ile Leu Lys Gln Leu Arg Thr 1 5 10 15 gca aaa ggg cta tcg cag gag caa cta gcc ctg agc tgt ggc ttg gac 96 Ala Lys Gly Leu Ser Gln Glu Gln Leu Ala Leu Ser Cys Gly Leu Asp 20 25 30 cgt aca ttt att tca atg tta gaa aga ggg caa agg cag ccg tct tta 144 Arg Thr Phe Ile Ser Met Leu Glu Arg Gly Gln Arg Gln Pro Ser Leu 35 40 45 tcg tct atc ctc tcc cta tca aaa tcg ctt gaa aca cct gcg cac gag 192 Ser Ser Ile Leu Ser Leu Ser Lys Ser Leu Glu Thr Pro Ala His Glu 50 55 60 atg cta aag aaa act acg gat tta ata gac tca gaa aaa tct taa 237 Met Leu Lys Lys Thr Thr Asp Leu Ile Asp Ser Glu Lys Ser 65 70 75 6 78 PRT Oceanospirillum kriegii 6 Met Lys Val Glu Cys Ala Phe Gly Arg Ile Leu Lys Gln Leu Arg Thr 1 5 10 15 Ala Lys Gly Leu Ser Gln Glu Gln Leu Ala Leu Ser Cys Gly Leu Asp 20 25 30 Arg Thr Phe Ile Ser Met Leu Glu Arg Gly Gln Arg Gln Pro Ser Leu 35 40 45 Ser Ser Ile Leu Ser Leu Ser Lys Ser Leu Glu Thr Pro Ala His Glu 50 55 60 Met Leu Lys Lys Thr Thr Asp Leu Ile Asp Ser Glu Lys Ser 65 70 75 7 24 DNA Synthetic 7 tgcattaaca ggacttcaat cacc 24 8 24 DNA synthetic 8 acataatgca ggccacgccc aacc 24 9 24 DNA synthetic 9 cggtctggat gaaaggaatt cggc 24 10 24 DNA synthetic 10 aatctttcaa atcgccgtag cact 24 11 24 DNA synthetic 11 tcctgtatga gacatcttca tcag 24 12 24 DNA synthetic 12 caccattacc tttgcgaaca ggat 24 13 42 DNA synthetic 13 ggaggagtcc atatgaaaat aaagcgtatt gaggtcctta ta 42 14 42 DNA synthetic 14 ggaggagtcg actcacctta tagcacgacc atctgtaccc tt 42 15 42 DNA syntheti 15 ggaggagtcc atatgaaaat aaagcgtatt gaggtcctta ta 42 16 27 DNA synthetic 16 ccttatagca cgaccatctg taccctt 27 

What is claimed is:
 1. Isolated DNA encoding the OkrAI restriction endonuclease, wherein the isolated DNA is obtainable from Oceanospirillum kriegii.
 2. A cloning vector comprising a vector into which a DNA segment encoding the OkrAI restriction endonuclease has been inserted.
 3. Isolated DNA encoding the OkrAI restriction endonuclease, wherein the isolated DNA is obtainable from ATCC No. PTA-5002.
 4. A cloning vector which comprises the isolated DNA of claims 1 or
 3. 5. A host cell transformed by the cloning vector of claims 2 or
 4. 6. A method of producing recombinant OkrAI restriction endonuclease comprising culturing a host cell transformed with the vector of claims 2 or 4 under conditions suitable for expression of said endonuclease. 